Molecular basis of insecticide resistance with special reference to

Bacillus thuringiensis

Presented by-Deba Jit DasPhD 3rd year, 1st semester

What does insecticide resistance mean?

Mechanism of development of resistance

Resistance against Bacillus thuringiensis

Strategies to delay resistance development against Bt

Genetic regulation of relative fitness in Cry1Ac resistant Helicoverpa

armigera larvae

Points to be discussed..

The decreased susceptibility of a pest population to

a pesticide that was previously effective at controlling

the pest

Or

The genetically acquired ability of an organism to

survive a pesticide application at doses that once

killed most individuals of the same species

What is insecticide resistance??

Phase I- Due to selection of variants in the population

according to genetic principle, the resistance which is

initially present in the population is expressed.

Phase II- Acceleration of resistance takes place by

induction of pre-existing detoxifying enzymes towards

enhanced activity, resulting in faster breakdown of the

chemicals.

Two phases of resistance

How does resistance occur in pest population??

I. Behavioral Resistance

Mechanisms of insecticide resistance…

Development of ability to avoid a dose that would prove lethal.

Stimulus dependent and matter of hypersensitivity or hyperirritability

Avoid lethal dose or treated surface

Behavioural resistance??

Physiological resistance

Reduced penetration

Target site insensitivity

Enhanced detoxification

Cuticle contains more protein and Lipid.

Increased sclerotization.

Binding protein and lipid reservoir traps

insecticide in the cuticle.

Slight resistance.

What is reduced penetrance??

Target site insensitivity

What are the different targets of different group of insecticides??

InsecticideDetoxification of insecticides by enzymes

Target site

Enhanced detoxification

Detoxifying Enzymes

a. Hydrolases –Carboxyl Esterase (CarE)

i. Esterase gene amplification

i i. Esterase mutation

b. Mixed fuction oxidases (MFO’s)

Cytochrome P450 monoxygenases (P450)

c. Glutathion-S-transferases (GST)

What are the different detoxifying enzymes??

General defense mechanism in insects against insecticides…

Resistance development against Bacillus

thuringiensis

Bacillus thuringiensis

Bacillus thuringiensis (Bt ) is a common gram positive,

spore-forming, soil bacterium.

In stress condition Bt undergo sporulation and synthesize

insecticidal crystal proteins (ICPs) or Cry proteins.

Benefits of Bt proteins

High specificity and potency,

Reduction in chemical pesticide applications

Increased crop yield.  

How does Cry protein act??

Mechanisms of resistance against Cry protein..

Four major mechanisms of resistance of insects against

Bt toxins are

Alteration in protease activity

Sequestration of the toxin by glycolipid moieties

Alteration in cry toxin receptor

Alteration in ABCC2 transporter

Schematic representation of different mechanism of resistance against Cry toxin

How does mutated ABCC2 transporter induce resistance in insects??

How can the toxicity of cry protein be enhanced??

Chitinase improves accessibility of the toxin to the epithelial

membrane.

Serine protease inhibitors reduce degradation of Cry or of

toxin-receptors.

Introduction of intramolecular cleavage sites

Expression of hybrid toxin

Deletion of helix -1 that induces toxin-oligomerization and α

skip cadherin interaction.

Sites for modification of Cry toxins…

Strategies to delay resistance development against Bt

Genetic regulation of relative

fitness in Cry1Ac resistant

Helicoverpa armigera larvae

Up-regulated death associated LIM-only protein contributes to fitness costs of Bacillus thuringiensis

Cry1Ac resistance in Helicoverpa armigeraHuiming Guo , Guoqing Lu , Xiaofeng Su , Gemei Liang , Chenxi Liu ,

Hongmei Cheng (2014)

Enhanced detoxificationBrief overview of the study….

cDNA-AFLP analysis was used to identify differentially

related gene both in susceptible and resistant insects

Ha-DALP gene was differentially expressed in resistant

Helicoverpa larvae

Ha-DALP gene is associated with transcriptioanal

regulation, cell signalling and cell fate determination

Enhanced detoxificationObjective of the study….

To analyze the expression pattern of Ha-DALP

gene in both Cry1Ac resistant (BtR) and

susceptible (96S) insects

To compare the fitness-trade off between two

groups of insects

Enhanced detoxificationExperimental procedures and findings

Two strains of H.armigera were used for the study

purpose

Cry1Ac resistant Bt strain (BtR)

Cry1Ac susceptible strain (96S)

Resistant insects were selected from the susceptible

strain reared for 75 generations in the presence of

Cry1Ac protoxin

Enhanced detoxificationRNA extraction and cDNA cloning

RNA was extracted from different stages (2 n d , 3 r d ,4 t h , and

5 t h instar) and tissues (epidermis, midgut and whole body)

RACE was performed to obtain the full lenth sequence of

HaDALP gene

Sequence comparison with Menduca sexta HaDALP gene

Multiple sequence alignment result

Ha-DALP gene expression at

different stage of larval

growth and in different

tissues was checked by qRT-

PCR.

96S and Bt-R Ha-DALP expression analysis

FISH was carried out to determine specific expression of Ha-DALP gene in different stages and tissues

of 96s and Bt-R insects.

Analysis of Ha-DALP expression by FISH

FISH analysis of HaDALP transcription levels in the 96S 2nd instar larvae

FISH analysis of HaDALP transcription levels in the Bt-R 2nd instar larvae

To express Ha-DALP specific dsRNA a recombinant

plasmid (RNAi-DALP) was constructed

Primers were designed to to amplify a 423 bp fragment

from Ha-DALP gene

RNAi-DALP plasmid was transformed into competent

HT115 cells

Bacteria containing Ha-DALP specific dsRNA were

harvested and used for H. armigera feeding bioassay

RNAi analysis in 96S larvae

qRT-PCR and RT-PCR analysis of Ha-DALP silencing

Enhanced detoxificationMeasurement of larval weight loss

Purified Ha-DALP protein feeding bioassay

Recombinant expression vector pET32a-HaDALP was

constructed using primers designed from Ha-DALP gene

sequence

HaDALP protein was isolated and purified by affinity

purification

Purified HaDALP protein used for feeding the

experimentel groups

Effect of feeding purified Ha-DALP protein to 96S larvae

Conclusion of the discussion

To overcome the selection pressure a large group insects has

developed resistance against insecticides as well as Bt

But there is enough score for us to enhance the efficacy of Cry toxin

Genetic change in insects resulting in Bt resistance can also affect

their fitness